This proposal is comprised of several projects aimed at developing new indicator dye and microscope imaging technologies, and projects designed to use fluorescent probes and quantitative imaging to explore cell biological and biophysical mechanisms. 1) We will continue the development of potential sensitive dyes by concentrating on: the synthesis of compounds for application in the near infrared region of the spectrum; covalent labeling to probe electrical activity at specific sites in a membrane; exploration of second harmonic generation as a high resolution, selective, and less invasive modality for monitoring electrical activity. 2) Convolution of computationally synthesized 3D objects with an experimental point spread function can be used to quantitatively assess fluorescence densities in confocal images; we plan to develop this idea with new techniques for modeling microscopic structures and validation of the accuracy of the intensity distributions. To improve resolution in optical microscopy, we will characterize and validate an inverse point spread function filter that restores out of focus light in 3D images; this filter is faster, less prone to artifact, and promises to provide better resolution than iterative deconvolution. 3) We have shown that intramembrane electric fields originating from dipole potentials or differences in surface potential, can vary in different regions of a cell and can alter the activity of voltage-sensitive channels. We will test the hypothesis that intracellular differences in intrinsic membrane electrical properties can correspondingly sensitize regions of N1E-115 neuroblastoma cells to differentially respond to stimuli. This will be achieved by combining electrophysiology and quantitative microscope imaging. 4) We will explore the physiology of mitochondria and endoplasmic reticulum during cell signaling. The research proposed here will focus primarily on how they respond during a complex cellular event; of particular interest will be how their membrane potentials, luminal pH, and luminal [Ca2+], might change in response to pHcyt and [Ca2+]cyt. The spatial and dynamic interactions between these organelles within the differentiated neuroblastoma cell will be probed experimentally and analyzed with image-based modeling and simulation.